Novel Benzamidine Derivatives Having Anti-Inflammatory and Immunosuppressive Activity

ABSTRACT

Compounds which can be represented by the general formula (I) indicated below: 
     
       
         
         
             
             
         
       
     
     and in which:
         A is selected independently from the carboxamide group, the thiocarboxamide group, and the carbonyl group,   R 1  is selected from an alkyl group having from 1 to 3 carbon atoms and the amino group, unsubstituted or substituted with the nitro group or the methyl group,   R 2  is selected independently from hydrogen, an alkyl group having from 1 to 4 carbon atoms, the methoxy, ethoxy, propoxy group, a mono-, bi- or tricyclic cycloalkane residue having from 5 to 12 carbon atoms, the adamantyl group, an aryl, naphthyl or heterocyclic group, unsubstituted or substituted with methyl, methoxy, hydroxy, amino or halogen groups,   R 3  and R 4  are selected independently from hydrogen and an alkyl group having from 1 to 3 carbon atoms,   R 5  represents one or two substituents independently selected from hydrogen and the methyl, methoxyl and hydroxyl groups,   n is a whole number from 0 to 6, and   the amidine group is in the para or meta position relative to the “-A-NH-” group.

This is a continuation of Application Ser. No. 11/652,675 filed Jan. 12,2007, which is a continuation of Application Ser. No. 10/467,624 filedJan. 6, 2004, which is a National Stage Application filed under §371 ofPCT Application No. PCT/EP02/01201 filed Feb. 6, 2002, which claimspriority from Italian Application No. T02001A000110, filed on Feb. 8,2001. The entire disclosure of the prior applications is considered partof the disclosure of the accompanying continuation application and ishereby incorporated by reference.

BACKGROUND OF THE INVENTION

The subject of the present invention is novel amidine derivatives ofphenylenediamine which can be represented by the general formula (I)indicated below:

and in which:

A is selected independently from the carboxamide group, thethiocarboxamide group, and the carbonyl group,

R₁ is selected from an alkyl group having from 1 to 3 carbon atoms andthe amino group, unsubstituted or substituted with the nitro group orthe methyl group,

R₂ is selected independently from hydrogen, an alkyl group having from 1to 4 carbon atoms, the methoxy, ethoxy, or propoxy group, a mono-, bi-or tricyclic cycloalkane residue having from 5 to 12 carbon atoms, theadamantyl group, an aryl, naphthyl or heterocyclic group, unsubstitutedor substituted with methyl, methoxy, hydroxy, amino or halogen groups,

R₃ and R₄ are selected independently from hydrogen and an alkyl grouphaving from 1 to 3 carbon atoms,

R₅ represents one or two substituents independently selected fromhydrogen and the methyl, methoxyl and hydroxyl groups,

n is a whole number from 0 to 6, and

the amidine group is in the para or meta position relative to the“-A-NH-” group.

In the compounds of the invention, R₂ is directly linked to the A group(n=o) or is linked to A through an alkilene group, having from 1 to 6carbon atoms, optionally substituted with one or more alkyl groupshaving from 1 to 3 carbon atoms.

SUMMARY OF THE INVENTION

The compounds of the present invention have been found to be potentantagonists of various mediators of inflammation and also haveimmunosuppressive properties. In vitro, they have been found to beinhibitors of inducible nitric oxide synthase (iNOS) and of the enzymecyclooxygenase (COX). In vivo, they have been found to be potentinhibitors of the cytokine “tumour necrosis factor” (TNFα). Moreover,many of the products of the invention can antagonise the collagenaseactivity of the metalloproteases.

Nitric oxide (NO) is formed at cell level by L-arginine, by means of theenzyme NOS. There are three subtypes of this enzyme. The enzyme (iNOS)which can be induced in the presence of pro-inflammatory cytokines or ofendotoxins is expressed in cells of many types, amongst which aremacrophages and neutrophiles.

Vasodilatation, which is a characteristic of acute inflammation,depends, for many mediators of the inflammatory process, such as, forexample, histamine, bradykinin, substance P, PAF, etc., on the releaseof NO. In general, NO increases the inflammatory responses in manyexperimental models, both acute and chronic.

It should be noted that NO can be produced massively in response to astimulus induced by cytokines in the inflamed joints of patients withrheumatoid arthritis and osteoarthritis, and that the plasmaconcentrations of NO in the sinovial fluid in these patients aregenerally very high.

The fact that the activity of iNOS is also very high in the colons ofpatients with ulcerative colitis is also interesting.

The prostaglandines (PGE) are mediators of inflammation generated by theenzyme cyclooxygenase (COX). The inducible isoform (COX-2) isoverproduced (“upregulated”) in the inflamed tissues and this leads toincreased synthesis of PGE.

There are interactions between the NOS and COX systems and the role ofNO in inflammation may therefore depend not only on its direct effect,but also on its modulatory effect on the bio-synthesis of PGE.

TNFα is a primary cytokine which initiates the cascade of events thatcharacterise an inflammatory process, inducing the synthesis and releaseof secondary cytokines and enzymes such as metalloproteases (MMP,amongst which is collagenase), iNOS and COX-2. As already mentioned, theintestinal mucosa is one of the most important sites of pro-inflammatorycytokine production, as observed in pathological conditions such aschronic inflammation of the colon (IBS) and ulcerative colitis.

It can therefore be considered that the compounds of the presentinvention may be used with advantage in the treatment of variousdiseases in man which are characterised by non-specific inflammationsuch as, for example, rheumatoid arthritis which is a syndrome with achronic course which can develop into progressive destruction of thejoint and periarticular structures, osteoarthrosis which is a diseasecharacterised by the degeneration of the joint cartilage, oftenaccompanied by secondary inflammation of the sinovial membrane, or inother pathological conditions, for example, in the gastrointestinalsystem, ulcerative colitis, Crohn's disease, IBS or food allergies andintolerance.

Advantageous use of the compounds of the invention can also be predictedin other areas and systems, for example, in the treatment ofpathological conditions of the cardiovascular system with aninflammatory or atherosclerotic basis which are sensitive to treatmentof iNOS inhibitors.

Moreover, for the compounds of the invention which have MMP-inhibitingactivity, advantageous use can be predicted in the treatment of tumoralconditions, since they potentially prevent localised or metastaticinvasion of tumoral cells, both by inhibiting the activation of variousgrowth factors and by blocking angiogenesis.

An enormous number of studies have been performed in the search fordrugs with anti-inflammatory activity which can perform an inhibitingaction on pro-inflammatory cytokines and which are free of the sideeffects of conventional anti-inflammatory drugs (COX inhibitors).

Chiou et al [Exp. Opin, Ther. Patents 6(1), 41-56 (1996)] have recentlyreviewed, in a monographic work, a large number of publications andpatents in which various classes of compounds which inhibit theproduction of cytokines by blocking their release, their receptors, ortheir converting enzymes, are described. Many monographic works havealso been published on the inhibitors of MMPs, such as, for example,that of Summers et al [Annual Reports in Med. Chemistry 33, 131-140(1998)] in which various chemical classes of MMP inhibitors are examinedand their therapeutic potential is discussed. Amongst others, patents inwhich the NO-synthase inhibitory activity of various amidines isdescribed, such as, for example, the patent PCT/GB/92/02387 and thepatent PCT/GB/94/01325, have been published.

However, the compounds described are amidine derivatives of amino-acidswhich are structurally very similar to analogous derivatives ofL-arginine, such as L-N-monomethyl arginine which is the subject of thepatent WO91/04024, but are different from the benzamidines of thepresent invention, both structurally and with regard to theirpharmacological activity as a whole.

All of these publications and researches show that there is a greattherapeutic need to find ever more potent and better-tolerated novelanti-inflammatory drugs. In accordance with this need, the object of thepresent invention is to provide, for treatment, novel drugs which,simultaneously, have anti-inflammatory and immunosuppressive activity,expressed by their combined iNOS and COX antagonistic activities, theirMMP-inhibiting activity, and their activity in inhibiting the productionof TNF-α, and which can thus be used advantageously in the treatment ofpathological conditions in man which are characterised by non-specificor autoimmune-based inflammation.

Pharmaceutical forms of the compounds of the invention can be preparedby conventional techniques, for example, as tablets, pills, capsules,suppositories, suspensions, solutions, patches, creams or ointments, andcan be administered by oral, parenteral, rectal, transdermal, ortransmucosal routes, or in other forms suitable for achieving thetherapeutic effect such as, for example, solid preparations for oral usewith delayed action which permit the controlled release of the activesubstance over time.

The active ingredient is usually administered to the patient with areference dose variable from 0.1 to 10 mg/kg of body weight per dose.

For parenteral administration, the use of a water-soluble salt of thecompounds of the invention such as the hydrochloride or another saltderived from a non-toxic and pharmaceutically-acceptable inorganic ororganic acid is preferred. For the derivatives of the invention with aslightly acid character, such as the derivatives in which R₁ is thenitro-amino group, the corresponding sodium salts or equivalent saltscan be prepared by conventional methods.

Substances commonly used in pharmaceuticals such as excipients, binders,flavourings, disaggregants, substances for stimulating transdermal andtransmucosal absorption, colourings, humectants, etc., may be used asinactive ingredients.

The method for the preparation of the derivatives of the inventionconsists of a series of reactions which comprises:

a) reacting the 1,3 or 1,4-phenylenediamine of formula (IV), suitablysubstituted and in which R₅ has the meaning given above, with theappropriate isothiocyanate (V A), acyl chloride (V B), or isocyanate (VC), in which R₂, R₃, R₄ and n have the meanings given above, in thepresence of an excess of phenylenediamine, in an inert solvent and at atemperature of between 4° C. and the reflux temperature of the solventused, to give the corresponding anilines of formula (III) in which R₂,R₃, R₄, R₅, A and n have the meanings given above, and in which theamine group is in the meta or para position relative to the chain withthe “A-NH” group (see General Synthesis Scheme, Step 1), and

b) reacting the anilines of formula (III), in which R₂, R₃, R₄, R₅, Aand n have the meanings given above, with the appropriate imidate offormula II, generally salified in hydrochloride form, in which R₁ hasthe meaning given above.

The reaction takes place in the presence of an excess of (II) relativeto (III) (preferably of 2 moles to 1) and in the presence of astoichiometric quantity, relative to (II), of a tertiary base,preferably triethylamine, in an inert anhydrous solvent, such as, forexample tetrahydrofuran, at a temperature of between 4° C. and theboiling point of the solvent, for a period of between 2 and 48 hours, togive the corresponding final derivatives of formula (I) in which A, R₁,R₂, R₃, R₄, R₅ and n have the meanings given above and in which theamidine group is in the para or meta position relative to the “A-NH”group. The compounds of formula (I) described in Table 2 (I-30 and I-31)were obtained with the use of reagents other than those of the generalformula (II) and, in particular, benzotriazolo-1-carboxamidinum tosylate(see Example 5) and N-methyl-N-nitroso-N′-nitroguanidine (see Example6), respectively.

The starting phenylenediamines, as well as the isothiocyanates (V A),the acyl chlorides (V B), the isocyanates (V C), and the imidates offormula (II) are commercially available or were prepared by conventionalmethods in accordance with existing literature.

The following examples are given below as further illustration of theinvention.

EXAMPLE 1 Preparation of N-(4-aminophenyl)-N′-pentyl Thiourea (CompoundIII-4 of Table 1)

71.8 g of 1,4-phenylenediamine (0.66 moles) was suspended in 300 ml oftetrahydrofuran, and 43 g of pentyl isothiocyanate (0.33 moles),dissolved in 50 ml of tetrahydrofuran, was added slowly dropwise, withstirring and at ambient temperature. After 24 hours, the solvent wasevaporated under vacuum and the residue, taken up with ethyl acetate,was washed with water, 0.1N citric acid, saturated sodium bicarbonate,and water to neutral pH. The solvent was rendered anhydrous withanhydrous sodium sulphate and evaporated under vacuum, to give 75 g ofcrude product which was re-crystallised from toluene. 60 g was obtained.

Formula: C₁₂H₁₉N₃S (M.W. 237.46). Yield 77%.

TLC: (chloroform/methanol 9/1) rf 0.7. M.P. 125° C.

HPLC: retention time (rt) 4.60 minutes.

HPLC conditions: Supelcosil LC-DP column, 100×4.6 mm, eluent KH₂PO₄0.01M 25/MetOH 27/MetCN 48 (pH 2.1), flow 0.4 ml/min, UV detector at 248nm.

¹HNMR (DMSO-d₆), ppm: 2.21 (bt, 3H, J=5.60 Hz); 0.98-1.71 (m, 6H); 3.33(q, 2H, J=6.68 Hz); 4.93 (bs, 2H); 6.45 (d, 2H, J=8.59 Hz); 6.81 (d, 2H,J=8.59 Hz); 7.05 (m, 1H); 8.88 (s, 1H).

All of the intermediate compounds of formula (III) according to theinvention in which A is the thiocarboxamide group were synthesised withthe use of the same method (see Scheme 1, step 1a).

EXAMPLE 2 Preparation of N-(4-aminophenyl)-N′-pentylamide (CompoundIII-22 of Table 1)

20.2 g di 1,4-phenylenediamine (0.181 moles) was dissolved in 200 ml oftetrahydrofuran, together with 11.1 ml of triethylamine (0.080 moles).The solution was cooled to 0° C. and 10 ml of caproyl chloride (0.0724moles) was added slowly dropwise so that the temperature did not exceed5° C. Upon completion of the addition, the temperature was increased toambient temperature. After reaction for 24 hours, the solvent wasevaporated under vacuum and the residue, taken up with ethyl acetate,was washed with water. The solvent was rendered anhydrous with anhydroussodium sulphate and evaporated under vacuum to give 12 g of crudeproduct which was recrystallised from toluene. 7.5 g was obtained.

Formula: C₁₂H₁₈N₂O (M-W. 206.28). Yield 51%;

TLC: (chloroform/methanol 9/1) rf 0.51. M.P. 88.5-89° C.

HPLC: retention time (rt) 5.54 minutes.

HPLC conditions: see Example 1.

¹HNMR (DMSO-d₆), ppm: 0.86 (bt, 3H, J=5.59 Hz); 1.03-1.79 (m, 6H); 2.18(t, 2H, J=6.99 Hz); 4.69 (s, 2H); 6.43 (d, 2H, J=8.39 Hz); 7.15 (d, 2H,J=8.39 Hz); 9.28 (s, 1H).

EXAMPLE 3 Preparation of N-(4-aminophenyl)-N′-cyclohexyl Urea (CompoundIII-28 of Table 1)

10.4 g of 1,4-phenylenediamine (0.095 moles) was suspended in 100 ml oftetrahydrofuran and 5 ml of cyclohexyl isocyanate (0.038 moles),dissolved in 20 ml of tetrahydrofuran, was added slowly dropwise, withstirring and at ambient temperature. After 24 hours, the solid formedwas filtered out and washed with cold tetrahydrofuran, water, and ethylether. 8.8 g was obtained.

Formula: C₁₃H₁₉N₃O (M.W. 233.31). Yield 98%

TLC: (chloroform/methanol 9/1) rf 0.40. M.P. 199.8-202.4° C.

HPLC: retention time (rt) 6.39 minutes.

HPLC conditions: see Example 1.

¹HNMR (DMSO-d₆), ppm: 0.75-2.00 (m, 10H); 3.47 (m, 1H); 4.62 (bs, 2H);5.75 (d, 2H, J=7.50 Hz); 6.49 (d, 2H, J=8.75 Hz); 7.00 (d, 2H, J=8.75Hz); 7.75 (bs, 1H).

Some derivatives of formula (III) obtained as described above are givenin Table 1 below, with some identifying chemical and physicalcharacteristics.

TABLE 1 Compounds of formula (III) (III)

M.P. (crystallisation Compound R₂ R₃ n A Crude Formula solvent)^(d) TLC(R_(f))^(e) III-1 CH₃ — 0 NH—CS C₈H₁₁N₃S 173.5-174.2 (A) 0.65 (I) III-2CH₃ H 2 NH—CS C₁₀H₁₅N₃S 118.6-120.2 0.58 (I) III-3 CH₃ H 3 NH—CSC₁₁H₁₇N₃S 124.5-127 0.67 (I) III-4 CH₃ H 4 NH—CS C₁₂H₁₉N₃S   113-115 (B)0.70 (I) III-5 CH₃ H 5 NH—CS C₁₃H₂₁N₃S 119.5-120.3 0.75 (I) III-6 CH₃ H6 NH—CS C₁₄H₂₃N₃S 107.0-107.4 0.75 (I) III-7 Isopropyl H 2 NH—CSC₁₂H₁₉N₃S 153.5-154.6 (B) 0.60 (I) III-8 CH₃ CH₃ 1 NH—CS C₁₀H₁₅N₃S137.2-137.8 (B) 0.63 (I) III-9 Ethyl CH₃ 1 NH—CS C₁₂H₁₉N₃S 165.6-166.80.75 (I) III-10 CH₃—O H 3 NH—CS C₁₁H₁₇N₃OS 104.6-105.2 0.65 (I) III-11Cyclohexyl — 0 NH—CS C₁₃H₁₉N₃S 168.6-169.4 0.70 (I) III-12 Phenyl — 0NH—CS C₁₃H₁₃N₃S 263.0-264.0 (A) 0.79 (II) III-13 Phenyl H 2 NH—CSC₁₅H₁₇N₃S 168.2-169.0 0.83 (II) III-14 Phenyl H 3 NH—CS C₁₆H₁₉N₃S117.3-117.9 0.83 (II) III-15 4-F-Phenyl H 2 NH—CS C₁₅H₁₇ClFN₃S190.2-191.6 0.85 (II) III-16 4-Cl-Phenyl H 2 NH—CS C₁₅H₁₆ClN₃S116.6-116.7 0.83 (II) III-17 2,6-diF-Phenyl H 2 NH—CS C₁₅H₁₅F₂N₃S147.7-149.7 0.50 (I) III-18 2-Piridyl H 1 NH—CS C₁₃H₁₄N₄S 106.0-108.00.41 (I) III-19 2-Piridyl H 2 NH—CS C₁₄H₁₆N₄S  82.0-84.0 0.38 (I) III-205-CH₃-2- — 0 NH—CS C₁₁H₁₂N₄S₂ 157.5-159.0 0.40 (I) Thiazolyl III-21 CH₃H 4 NH—CS C₁₄H₂₃N₃S 101.4-102.8 0.75 (I) III-22 CH₃ H 4 CO C₁₂H₁₈N₂O 88.5-89.0 (B) 0.51 (I) III-23 Cyclohexyl — 0 CO C₁₃H₁₈N₂O 176.1-177.0(B) 0.50 (I) III-24 1-Adamantyl — 0 CO C₁₇H₂₂N₂O 169.6-171.2 0.45 (I)III-25 2-Indolyl — 0 CO C₁₅H₁₃N₃O 193.8-194.9 (A) 0.47 (I) III-263-Indolyl H 1 CO C₁₆H₁₄N₃O 122.0-122.7 (B) 0.40 (I) III-271-CH₃-2-Indolyl — 0 CO C₁₆H₁₅N₃O 232.6-235.3 0.55 (I) III-28 Cyclohexyl— 0 NH—CO C₁₃H₁₉N₃O 199.8-202.4 0.40 (I) III-29 Cyclohexyl — 0 NH—CSC₁₃H₁₉N₃S 101.0-102.8 0.70 (I) Note: ^(a)In all of the compounds givenby way of example, R₄ is H, with the exception of compound III-9 inwhich R₄ is CH₃. ^(b)In all of the compounds given by way of example,the amino group is in the para position relative to the “NH—A” group,with the exception of compound III-29 in which the amino group is in themeta position relative to the “NH—A” group. ^(c)In all of the compoundsgiven by way of example, R₅ is H except for compound III-21 in which R₅is 2,5-dimethyl. ^(d)Crystallisation solvent: A (isopropanol); B(toluene). ^(e)Eluent: (I) chloroform/methanol (9/1) (v/v); (II)chloroform/methanol/water/ammonia (85/25/2/1) (v/v).

EXAMPLE 4 Preparation of N-[4-(N-acetamidine)phenyl]-N′-pentyl Thiourea(Compound I-4 of Table 2)

55 g di N-(4-aminophenyl)-N′-pentyl thiourea (0.23 moles) was dissolvedin 300 ml of tetrahydrofuran. 64.5 ml of triethylamine (0.46 moles) and50.7 g of methyl acetimidate hydrochloride (0.46 moles) was added, withstirring at ambient temperature; the pH of the suspension wasapproximately 9. After 24 hours (the pH fell to 7), the solid wasfiltered out and washed with a little tetrahydrofuran and ethyl ether.The residue, taken up with water, was rendered basic with 4N sodiumhydroxide to pH 11 and was left for 1 hour with stirring and thenfiltered, washed with water, and ethyl ether and purified hot withacetonitrile. 51 g was obtained.

Formula: C₁₄H₂₂N₄S (M.W. 278.42). Yield 80%.

TLC: (butanol/acetic acid/water 5/2/2) rf 0.70; (chloroform/methanolsaturated with ammonia 9/1) rf 0.37. M.P. 191.4° C.

HPLC: retention time (rt) 7.70 minutes.

HPLC conditions: see Example 1.

¹HNMR (DMSO-d₆), ppm: 0.88 (bt, 3H, J=5.60 Hz); 1.00-1.55 (m, 6H); 1.82(s, 3H); 3.35 (m, 2H); 5.91 (bs, 1H); 6.63 (d, 2H, J=8.56 Hz); 7.11 (d,2H, J=8.56 Hz); 7.28 (bs, 1H); 9.08 (bs, 1H).

All of the derivatives of formula (I) in which R1 was methyl wereprepared in similar manner with the use of the appropriate aniline offormula (III) in place of N-(4-aminophenyl)-N′-pentyl thiourea.

EXAMPLE 5 Preparation of 1-guanidinophenyl-4-cyclohexyl Thiourea(Compound I-30 of Table 2)

15 g of N-(4-aminophenyl)-N′-cyclohexyl thiourea (0.06 moles) wassuspended in 100 ml of acetonitrile, and 20 g ofbenzotriazolo-1-carboxamidinium tosylate [0.06 moles, Katrizky, A. R. etal. Synth. Comm. 25(8), 1173-1186, (1995)] was added, with stirring atambient temperature. After 72 hours, the solvent was evaporated undervacuum and the residue, taken up with ethyl acetate, was washed with0.1N sodium hydroxide and extracted with 0.1N citric acid. The aqueousphase was brought to pH 9 with 2N sodium hydroxide, and extracted withethyl acetate and the organic phase was washed with water. The solventwas rendered anhydrous over anhydrous sodium sulphate and evaporatedunder vacuum to give 8 g of crude product which was purified withisopropyl ether. 7.4 g was obtained.

Formula: C₁₄H₂₁N₅S (M.W. 291.42). Yield 43%.

TLC: (butanol/acetic acid/water 5/2/2) rf 0.71. M.P. 188.6° C.

HPLC: retention time (rt) 8.0 minutes.

HPLC conditions: see Example 1.

¹HNMR (DMSO-d₆), ppm: 0.67-2.17 (m, 10H); 4.05 (m, 1H); 5.73 (bm, 6H);6.71 (d, 2H, J=8.43 Hz); 7.17 (d, 2H, J=8.43 Hz).

EXAMPLE 6 Preparation of 1-nitroguanidinophenyl-4-cyclohexyl Thiourea(Compound I-31 of Table 2)

9 g of N-(4-aminophenyl)-N′-cyclohexyl thiourea (0.036 moles) wassuspended in 140 ml of a 1/1 ethanol/water mixture, and 3 g ofN-methyl-N-nitroso-N′-nitroguanidine [0.021 moles, McKay, A. F. J. Am.Chem. Soc. 71, 1968-1970, (1949)] was added, with stirring at ambienttemperature. After 2 hours at ambient temperature, the reaction mixturewas heated under reflux for 1 hour; the precipitate which formed wasfiltered hot, washed with ethyl ether and dried. 3.8 g was obtained.

Formula: C₁₄H₂₀N₆O₂S (M.W. 336.41). Yield 54%.

TLC: (butanol/acetic acid/water 5/2/2) rf 0.95. M.P. 215.2° C.

HPLC: retention time (rt) 4.16 minutes.

HPLC conditions: see Example 1.

¹HNMR (DMSO-d₆), ppm: 0.84-1.99 (m, 10H); 4.01 (m, 1H); 7.15 (d, 2H,J=8.76 Hz); 7.42 (d, 2H, J=8.76 Hz); 7.52 (bd, 1H, J=7.64 Hz); 8.03 (bs,2H); 9.28 (s, 1H); 9.42 (bs, 1H).

EXAMPLE 7 Preparation of N-[4-(N-acetamidino)phenyl]-N′-pentyl ThioureaHydrochloride (the Hydrochloride of Compound I-4)

3 g of N-[4-(N-acetamidino)phenyl]-N′-pentyl thiourea (0.011 moles) wasdissolved in 1N HCl. After 0.5 hours at ambient temperature, theprecipitate which formed was filtered out, washed with a little water,and with ethyl ether, and dried. 3.2 g was obtained.

Formula: C₁₄H₂₃ClN₄S (M.W. 314.88). Yield 93%.

TLC: (butanol/acetic acid/water 5/2/2) rf 0.70. M.P. 180.6° C.

¹HNMR (DMSO-d₆), ppm: 0.84 (t, 3H, J=5.60 Hz); 1.02-1.78 (m, 6H); 2.28(s, 3H); 3.43 (bq, 2H, J=6.05 Hz); 7.15 (d, 2H, J=8.56 Hz); 7.73 (d, 2H,J=8.56 Hz); 8.39 (m, 1H); 9.42 (bs, 1H); 10.38 (s, 1H); 11.28 (s, 1H).

Some derivatives of formula (I) obtained according to the invention aregiven in Table 2 below, with some identifying chemical and physicalcharacteristics, without thereby in any way limiting the spirit or thescope of the invention.

TABLE 2 Compounds of formula (I) (I)

M.P. Com- (crystallisation TLC pound R₁ R₂ R₃ n A Crude formulasolvent)^(d) (R_(f))^(e) I-1 CH₃ CH₃ — 0 NH—CS C₁₀H₁₄N₄S 186.2-187.80.50 I-2 CH₃ CH₃ H 2 NH—CS C₁₂H₁₈N₄S 188.1-189.1 (A) 0.70 I-3 CH₃ CH₃ H3 NH—CS C₁₃H₂₀N₄S 182.3-183.1 (A) 0.72 I-4 CH₃ CH₃ H 4 NH—CS C₁₄H₂₂N₄S190.7-191.4 (A) 0.71 I-5 CH₃ CH₃ H 5 NH—CS C₁₅H₂₄N₄S 177.6-178.0 0.80I-6 CH₃ CH₃ H 6 NH—CS C₁₆H₂₆N₄S 177.8-178.4 0.80 I-7 CH₃ Isopropyl H 2NH—CS C₁₄H₂₂N₄S 177.7-179.1 (A) 0.58 I-8 CH₃ CH₃ CH₃ 1 NH—CS C₁₂H₁₈N₄S163.0-163.9 (A) 0.58 I-9 CH₃ Ethyl CH₃ 1 NH—CS C₁₄H₂₂N₄S 145.7-147.4 (B)0.61 I-10 CH₃ CH₃—O H 3 NH—CS C₁₃H₂₄N₄OS 146.9-149.6 (B) 0.54 I-11 CH₃Cyclohexyl — 0 NH—CS C₁₅H₂₂N₄S 171.3-171.6 (A) 0.60 I-12 EthylCyclohexyl — 0 NH—CS C₁₆H₂₄N₄S 155.0-156.0 0.66 I-13 CH₃ Phenyl H 0NH—CS C₁₅H₁₆N₄S 118.7-120.5 0.70 I-14 CH₃ Phenyl H 2 NH—CS C₁₇H₂₀N₄S186.6-188 0.63 I-15 CH₃ Phenyl H 3 NH—CS C₁₈H₂₂N₄S 146.6-148.0 (B) 0.60I-16 CH₃ 4-F-Phenyl H 2 NH—CS C₁₇H₁₉FN₄S 179.1-181.6 0.57 I-17 CH₃4-Cl-Phenyl H 2 NH—CS C₁₇H₁₉ClN₄S 174.0-176.0 (A) 0.60 I-18 CH₃2,6-diF-Phenyl H 2 NH—CS C₁₇H₁₈F₂N₄S 158.8-160.4 0.58 I-19 CH₃ 2-PiridylH 1 NH—CS C₁₅H₁₇N₅S 159.0-161.0 (A) 0.48 I-20 CH₃ 2-Piridyl H 2 NH—CSC₁₆H₁₉N₅S 171.5-173.0 (A) 0.42 I-21 CH₃ 5-CH₃-2-Thiazolyl — 0 NH—CSC₁₃H₁₅N₅S₂ 107.8-110.1 0.45 I-22 CH₃ CH₃ H 4 NH—CS C₁₆H₂₆N₄S   118-121.7(B) 0.61 I-23 CH₃ CH₃ H 4 CO C₁₄H₂₁N₃O 160.4-161.6 0.60 I-24 CH₃Cyclohexyl — 0 CO C₁₅H₂₁N₃O 194.6-195.8 (B) 0.60 I-25 CH₃ 1-Adamantyl —0 CO C₁₉H₂₅N₃O 224.4-224.7 0.61 I-26 CH₃ 2-Indolyl — 0 CO C₁₇H₁₆N₄O192.0-193.5 0.61 I-27 CH₃ 3-Indolyl H 1 CO C₁₈H₁₈N₄O 159.0-160.0 0.51I-28 CH₃ 1-CH₃-2-Indolyl — 0 CO C₁₈H₁₈N₄O 195.5-196.5 0.68 I-29 CH₃Cyclohexyl — 0 NH—CO C₁₅H₂₂N₄O 220.2-224.6 0.50 I-30 NH₂ Cyclohexyl — 0NH—CS C₁₄H₂₁N₅S 176.5-179.4 0.71 I-31 NO₂—NH Cyclohexyl — 0 NH—CSC₁₄H₂₀N₆O₂S 213.9-215.2 0.95 I-32 CH₃ Cyclohexyl — 0 NH—CS C₁₅H₂₂N₄S171.4-173.2 0.66 Note: ^(a)In all of the compounds given by example, R₄is H, with the exception of compound I-9 in which R₄ is CH₃. ^(b)In allof the compounds given by way of example, the amino group is in the paraposition relative to the “NH—A” group, with the exception of compoundI-32 in which the amino group is in the meta position relative to the“NH—A” group. ^(c)In all of the compounds given by way of example, R₅ isH, except for compound I-22 in which R₅ is 2,5-dimethyl.^(d)Crystallisation solvent: A (acetonitrile); B (toluene). ^(e)Eluent:butanol/acetic acid/water (5/2/2) (v/v).

Pharmacological Activity

a) The activity in inhibiting the formation of NO, measured as NO₂⁻(nitrites), PGE₂, and neutral protease, was investigated in vitro onculture broths of rabbit joint chondrocytes stimulated by cytokine IL-1β(1 ng/ml) for 48 hours. For the preparation of the chondrocytes, themethod described by Berenbaum et al [FEBS Letters 340, 51-55 (1994)] wasfollowed. Briefly, fragments of cartilage removed under sterileconditions from the heads of rabbit shoulder, hip and knee joints werechopped finely and digested at 37° C. by hyaluronidase, trypsin andcollagenase solutions, giving rise, after filtration on sterile gauzeand centrifuging at 600×g and suitable dilution with 10% DMEM-FCS, to aconcentration of approximately 1×10⁵ cells per well. The cells were keptin these conditions until confluence (about 15 days), the broth beingchanged every 3 days. At this point, the products under test, dissolvedin the medium, were added to each test sample and, after 20 minutes, 350μl IL-1β was added in order to have a final concentration of 1 ng/ml.The duration of the stimulation was 48 hours at 37° C. (incubationair-CO₂ 7%). Measurement of the nitrites, as described by Green et al.[Anal. Biochem. 126, 131-138 (1982)], and of the PGE₂s by RIAmeasurement, was then performed on the cell supernatant fluid. Themeasurement of the neutral proteases was performed in the cellsupernatant fluid containing the p-aminophenyl mercury acetate (APMA)activator with the use of azocoll as the substrate and with incubationat 37° C. for 17 h as described by Chavira et al. [Anal. Biochem. 136,446-450 (1984)]. In order to evaluate the direct inhibitory effect ofthe compounds under test on the hydrolytic activity of the cellsupernatant fluid, they were added to the supernatant fluid containingthe proteases induced by IL-1β and already activated by AMPA.

The results obtained are shown in Table 3, in which the IC₅₀, that is,the concentration (micromolar) of antagonist which can inhibit theformation of nitrites, PGE₂s, and neutral proteases, respectively, by50% relative to the control group, that is, to the cells stimulated withIL-1β but without the addition of antagonists, is given for some of thecompounds of the invention already given by way of example in Table 2.

TABLE 3 Compounds of formula (I) (I)

Rabbit joint chondrocytes NO PGE2 MP Compound R₁ R₂ R₃ n A IC₅₀(×10⁻⁶M)I-1 CH₃ CH₃ — 0 NH—CS IN IN 30 I-2 CH₃ CH₃ H 2 NH—CS IN IN 30 I-3 CH₃CH₃ H 3 NH—CS 190 250 13.3 I-4 CH₃ CH₃ H 4 NH—CS 6.6 3.3 6.6 I-5 CH₃ CH₃H 5 NH—CS 10.0 10.0 3.3 I-6 CH₃ CH₃ H 6 NH—CS 30.0 20.0 20.0 I-7 CH₃Isopropyl H 2 NH—CS 3.3 6.6 13.3 I-8 CH₃ CH₃ CH₃ 1 NH—CS 30.0 3.3 3.3I-9 CH₃ C₂H₅ CH₃ 1 NH—CS 10.0 10.0 6.6 I-10 CH₃ CH₃—O H 3 NH—CS 50.050.0 6.6 I-11 CH₃ Cyclohexyl — 0 NH—CS 110 16.6 30 I-12 Ethyl Cyclohexyl— 0 NH—CS IN 13.3 100 I-13 CH₃ Phenyl H 0 NH—CS 100 10 6.6 I-14 CH₃Phenyl H 2 NH—CS 100 33 6.6 I-15 CH₃ Phenyl H 3 NH—CS 33 16.6 6.6 I-16CH₃ 4-F-Phenyl H 2 NH—CS 13.3 26.6 6.6 I-17 CH₃ 4-Cl-Phenyl H 2 NH—CS10.0 10 10 I-18 CH₃ 2,6-diF-Phenyl H 2 NH—CS IN 6.6 6.6 I-19 CH₃2-Piridyl H 1 NH—CS 300 IN 13.3 I-20 CH₃ 2-Piridyl H 2 NH—CS 16.6 16.66.6 I-21 CH₃ 5-CH₃-2-Thiazolyl — 0 NH—CS 66.6 IN IN I-22 CH₃ CH₃ H 4NH—CS 16.6 10.0 20.0 I-23 CH₃ CH₃ H 4 CO 100 IN IN I-24 CH₃ Cyclohexyl —0 CO 33.3 33.3 IN I-25 CH₃ 1-Adamantyl — 0 CO IN 16.6 IN I-26 CH₃2-Indolyl — 0 CO 300 IN IN I-27 CH₃ 3-Indolyl H 1 CO 10 IN IN I-28 CH₃1-CH₃-2-Indolyl — 0 CO IN 100 IN I-29 CH₃ Cyclohexyl — 0 NH—CO 25.0 6.625.0 I-30 NH₂ Cyclohexyl — 0 NH—CS 6.6 10 13.3 I-31 NO₂—NH Cyclohexyl —0 NH—CS 46.6 30 6.6 I-32 CH₃ Cyclohexyl — 0 NH—CS 300 300 IN L-NAME — —— — — — IN 3(mM) IN Note: a) for the structural identification, seeNotes a, b and c of Table 2 b) NO determined as nitrites c) MP:metalloproteases

It can be seen from the data given in Table 3 that some of the compoundswhich were tested and which are subjects of the invention have a potentinhibitory effect, at micromolar level, on the production of nitrites,PGE₂s and metalloproteases induced by IL-1β cytokine in rabbitchondrocyte cultures. The best compounds were those in which R₁ was CH₃,R₃ and R₄ were H, R₂ was CH₃ if n was 4 or 5, isopropyl if n was 2, orthe 2,6-difluoro-phenyl group if n was 2, and in which A was NH—CS(compounds I-4, I-5, I-7 and I-18, respectively). Compound I-31 in whichR₁ was the NH—NO₂ group, R₂ was cyclohexyl, and A was the NH—CS groupwas also very active. It is interesting to note that the inhibitoryactivity on the metalloproteases was expressed solely by the compoundsin which A was the NH—CS group. It should also be noted that thereference NO-synthase inhibitor compound L-NAME generally had anactivity about 30-100 times less potent than the best compounds of theinvention, such as the compound I-4, and was completely inactive ininhibiting the metalloproteases.

b) Some of the compounds of the invention, such as compounds I-4, I-11and I-31, were evaluated in vivo in a series of experimentalextravasation models in which 5 μl of the preselected phlogogenic agentdissolved in physiological solution was injected intradermally into theears of mice as described by Erdo et al., with slight modifications[Agents and Actions 39, 137-142 (1993)].

The products under test were administered orally 1 hour before thechallenge and, 30 minutes before the challenge, the dye Evans Blu wasinjected intravenously in a dose of 100 mg/kg. The animals were killedat a time predetermined according to the test, 30 min.—2 hours after thechallenge. The extravasation was evaluated by determining the quantityof dye present in the ear, extracted after homogenisation of the tissuein 2 ml of formamide and incubation at 50° C. for 2 hours. Aftercentrifuging, the amount of dye was determined by measuring theabsorption at 620 nm. The maximum % effect (% MPE) was calculated by thefollowing formula:

${\% \mspace{14mu} {MPE}} = {\frac{\left( {E_{V} - E_{D}} \right)}{E_{V} - E_{B}} \times \; 100}$

in which E_(V) is the mean absorption observed in the group of animalstreated solely with the phlogogenic agent, E_(D) is the group treatedwith the phlogogenic agent and the drug, and E_(B) is the base value,that is, the value for the animals injected with physiological solutionalone.

The phlogogenic agents used were:

Arachidonic acid (dissolved in EtOH); (1 mg/mouse; killed+30 min);histamine (3 nmoli/mouse; killed+30 min); PAF (30 pmoli/mouse; killed+60min); Zymosan (10 μg/mouse; killed+120 min); bradykinin (0.6nmoli/mouse; killed+30 min).

The results obtained with Compound I-4 are summarised in Table 4.

TABLE 4 Activity of Compound I-4 on extravasation induced by algogenicagents in the ears of mice % inhibition effects Arachidonic Hista-Brady- Acid mine PAF Zymosan kinin Duration Dose mg/kg (OS) 30 min 30min 1 h 2 h 30 min 2.5 mg/kg — — 41.9 40.0 — 5 mg/kg 37.8 17.8 49.9 66.630.1 10 mg/kg 40.0 27.7 55.1 65.8 30.7 20 mg/kg 52.6 38.8 56.4 83.9 49.140 mg/kg 55.5 52.5 66.2 89.8 51.3 80 mg/kg — — — 95.5 —

Compound I-4 was found to be particularly effective in extravasationinduced by Zymosan (ED₅₀ 3.1 mg/kg); however, in the other extravasationmodels investigated, a dose of only 5 mg/kg also produced a meaninhibitory effect of approximately 35%. A non-selective NO-synthaseinhibitor, N-nitro-L-arginine methyl ester (L-NAME), a COX₁ inhibitor(Piroxicam) and a COX₂ inhibitor (Nimesulide) were used as comparisondrugs.

The results obtained are given in Table 5 below.

TABLE 5 Inhibition of extravasation in the ears of mice induced byalgogenic agents (ED₅₀ mg/kg OS) Hista- AA mine PAF Zymosan BradykininCompound I-4 20.5 37.0 6.1 3.1 32.6 Compound I-31 30.5 — 10.8 10.5 —L-NAME 55.1 36.4 40.0 IN (>100) 39.4 Piroxicam — — 21.2 6.1 — Nimesulide41.0 — IN (>40) — — Note: AA = Arachidonic acid (—): not tested

In general, Compound I-4 was the most active of the compoundsinvestigated. In fact, of the reference compounds, L-NAME wasapproximately as active as Compound I-4 in extravasation induced byhistamine and bradykinin but was two times less active in extravasationby AA, much less active in antagonising PAF, and completely inactive inextravasation induced by Zymosan.

Piroxicam was 2-3 times less active than Compound I-4 in the models inwhich PAF and Zymosan were used, and Nimesulide was less active (2times) in antagonising AA and was inactive in antagonising PAF. CompoundI-31 had an activity profile similar to that of compound I-4 but wasgenerally 2-3 times less active.

c) The immunosuppressive activity of Compound I-4 was evaluated in an invivo test in the rat, in which a lipopolysaccharide (LPS) of bacterialorigin, injected i.p. at a dose of 6 mg/kg, induced a shockcharacterised by urgent diarrhoea accompanied by a large increase in theplasma TNFα concentration.

Blood was taken from the animals which were killed 90 minutes after thechallenge and the concentration of TNFα in the plasma was determined byElisa (Amersham Kit cod. RPN2734). The results thus obtained are givenin Table 6.

TABLE 6 Effects of Compound I-4 on plasma TNFα concentration in ratsafter stimulation with LPS (6 mg/kg/I.P.) Inhibition TNFα (ng/ml ± SD)(%) Control (SHAM) <1 (n = 4) — Control LPS 61 ± 13.4 (n = 8) — CompoundI-4 2.3 ± 1.2 (n = 6) 96.2 (50 μg/kg ICV) + LPS Compound I-4 12.9 ± 7.2(n = 6) 78.9 (10 mg/kg IV) + LPS Compound I-4 30.6 ± 1.0 (n = 6) 49.8(20 mg/kg OS) + LPS

It is clear from the data given above that compound I-4 is very activein inhibiting the increase in plasma TNFα in the course of endotoxicshock induced by LPS. For example, the oral dose of 20 mg/kg inhibitedthe effect of the LPS by about 50%.

d) Intestinal anti-inflammatory activity.

TNFΔ is a cytokine implicated in the pathogenesis of a variety ofimmunology-based inflammatory diseases, amongst which is inflammation ofthe intestine. It has been shown [Bertrand et al. Br. J. Pharmacol. 124,1385-1394 (1998)] that inducing an overproduction of TNFα, induced byCOX₁-type anti-inflammatory drugs, brings about activation of theneutrophiles and an increase in the production of nitrites due to theactivation of the tissue iNOS, which together contribute to thetoxic-ulcerative effects on the intestinal mucosa.

The combined capability of some of the compounds of the invention toinhibit both iNOS and the synthesis of TNFα has led to their activitybeing checked in an experimental model of colitis caused by a chemicalhapten, trinitrobenzene-sulphonic acid (TNBS), which can bind to thetissue proteins and stimulate cell-mediated immunity. The intrarectaladministration of TNBS in association with ethanol causes acuteinflammation characterised by extensive ulceration and necrosis.

A distal colitis was therefore induced in the rat by intrarectalinstillation of TNBS (40 mg/kg) dissolved in 50% ethanol (0.5 ml/rat).The drugs were administered orally twice a day on days −2; −1; 0; +1 andthe animals were killed 48 hours after the administration of TNBS.

The parameters examined were: total weight of the colon (g), macroscopictissue damage “score”, measurement of the tissue myeloperoxidase (MPO)activity, which is a marker of the infiltration of the neutrophiles, andmeasurement of the iNOS activity (pmoles/g tissue/min.).

The macroscopic score (MDS) was taken on about 10 cm of colon inaccordance with the following arbitrary scale:

0 No damage 1 Hyperaemia (no ulcers) 2 1 small ulcer or erosion 3 1ulcer with inflammation 4 Two ulceration sites 5 More than twoulceration sites or 1 ulcer >1 cm 6-10 If the damage was >2 cm, thescore was increased by 1 for each cm

The tissue MPO measurement was performed according to Velgara et al.,JPET 1994. The measurement of the tissue nitrites was performed byapplying the method described above for the chondrocytes to thesupernatant fluid of the tissue homogenate.

Compound I-4, administered in doses of 5, 10 and 20 mg/kg, and compoundI-11 were examined, by administering them orally, in comparison withsulphasalazine (250 mg/kg) and 5-aminosalicylic acid (5-ASA) (100mg/kg), 2 drugs which are widely used in the treatment of ulcerativecolitis.

The results thus obtained are given in the following table.

TABLE 7 Effects of compound I-4, I-11, sulphasalazine and 5-ASA oncolitis induced by TNBS in rats Treatment groups Weight of the colonMacroscopic damage Myeloperoxidase activity iNOS activity [mg/kg; number(n)] (g) (MDS) (UMPO/g/min) (pmoles/g/min) Control (SHAM) (n = 6) 1.5 ±0.2 — 0.9 ± 0.3  6.6 ± 4.4 Control (TNBS) (n = 8) 2.4 ± 0.3 7.4 ± 1.47.7 ± 1.1 190.9 ± 98.6 I-4 (5 mg/kg; n = 8) 2.2 ± 0.2 5.0 ± 0.9 5.5 ±1.4 131.7 ± 91.9 I-4 (10 mg/kg; n = 8) 2.1 ± 0.3 4.4 ± 0.8(*) 5.0 ±1.7(*)  98.6 ± 60.0 I-4 (20 mg/kg; n = 8) 2.0 ± 0.2(*) 3.5 ± 0.8(*) 5.1± 1.2(*)  61.5 ± 31.4(*) I-11 (20 mg/kg; n = 7) 2.2 ± 0.3 4.3 ± 0.9(*)6.5 ± 1.8  90.6 ± 45.6(*) 5-ASA (100 mg/kg; n = 7) 2.4 ± 0.4 6.9 ± 1.17.9 ± 2.3 167.3 ± 106.5 Sulphasalazine (250 mg/kg; n = 7) 2.5 ± 0.3 6.0± 2.9 7.5 ± 5.5 100.8 ± 86.8 The values given are means ± StandardDeviation (*)value significantly different from the TNBS control group(Duncan's test)

It is clear from the data given in the table that compound I-4 had astrong protective effect in the experimental model of colitis induced inthe rat by TNBS. In fact I-4 reduces both macroscopic damage and all ofthe other inflammatory parameters taken into consideration in adose-dependent and significant manner at medium and high dose (10 and 20mg/kg). In particular, it reduces the increase in the weight of thecolon induced by treatment with TNBS, reduces macroscopic damage byabout 50% at doses of 10 and 20 mg/kg, and reduces the increase intissue MPO activity and iNOS activity induced by TNBS by 40% and 50%,respectively, at the same doses. In contrast, of the two reference drugsselected, 5-ASA (100 mg/kg) was inactive on all of the parameters, andsulphasalazine was slightly active at the very high dose of 250 mg/kgand in a non-statistically significant manner solely on macroscopicdamage (about 20% effect) and on iNOS activity (about 50% effect). Theother compound of the invention which was tested (Compound I-11) wasalso active at the dose of 20 mg/kg although the effects in reducingdamage, on the parameters of increase of the weight of the colon, andMPO activity were less clear than those of the compound I-4.

Finally, in an experimental model of colitis in the rat which imitatesas closely as possible a pathological condition which can be correlatedwith ulcerative colitis in man [Morris et al. Gastroenterology 96,795-803, (1989)] some of the compounds of the invention have aprotective effect much greater, and at lower doses, than that ofsulphasalazine which is a drug widely used in the treatment ofulcerative colitis and Crohn's disease.

1. A compound of formula (I) below:

wherein A is selected independently from a carboxamide group, athiocarboxamide group, and a carbonyl group; R₁ is selected from analkyl group having from 1 to 3 carbon atoms and an amino groupsubstituted with a nitro group when A is the thiocarboxamide group; R₁is selected from an alkyl group having from 1 to 3 carbon atoms when Ais selected independently from a carboxamide group and a carbonyl group;R₂ is selected independently from hydrogen, an alkyl group having from 1to 4 carbon atoms, a methoxy group, an ethoxy group, or a propoxy group,a mono-, bi- or tricyclic cycloalkane group having from 5 to 12 carbonatoms, an adamantyl group, and an aryl, naphthyl or heterocyclic group,unsubstituted or substituted with a methyl group, a methoxy group, ahydroxy group, an amino group or a halogen group; in which A, R₁, R₂,R₃, R₄, R₅ and n have the meanings given above and in which the amidinegroup is in the para or meta position relative to the -A-NH-group,recovering the compound of formula (I) or a pharmaceutically-acceptablesalt thereof from the reaction mass and purifying; c) alternatively, ifR₁ is NH—NO₂, the corresponding compound of formula (I) in which A isthe thiocarboxamide group and R₂, R₃, R₄, R₅ and n have the meaningsgiven above is prepared by reacting the anilines of formula (III) withN-methyl-N-nitroso-N′-nitroguanidine. R₃ and R₄ are selectedindependently from hydrogen and an alkyl group having from 1 to 3 carbonatoms; R₅ represents one or two substituents independently selected fromhydrogen and a methyl group, a methoxyl group and a hydroxyl group; n isa whole number from 0 to 6; and an amidine group is in the para or metaposition relative to the -A-NH-group, or a pharmaceutically acceptablesalt thereof.
 2. The compound of according to claim 1, wherein A is athiocarboxamide group; R₁ is an alkyl group having from 1 to 3 carbonatoms; R₂ is a methyl group or a mono-, bi- or tricyclic cycloalkanegroup having from 5 to 12 carbon atoms; R₃, R₄ and R₅ are hydrogen; n isa whole number between 0 and 6; and the amidine group is in the paraposition relative to the -A-NH-group, or a pharmaceutically acceptablesalt thereof.
 3. The compound according to claim 1 wherein A is selectedindependently from a carboxamide group and a carbonyl group; R₁ is analkyl group having from 1 to 3 carbon atoms; R₂ is a methyl group or amono-, bi- or tricyclic cycloalkane group having from 5 to 12 carbonatoms; R₃, R₄ and R₅ are hydrogen; n is a whole number between 0 and 6;and the amidine group is in the para position relative to the-A-NH-group, or a pharmaceutically acceptable salt thereof.
 4. Thecompound according to claim 1 wherein A is a thiocarboxamide group; R₁is selected from an amino group substituted with a nitro group; R₂ is amethyl group or a mono-, bi- or tricyclic cycloalkane group having from5 to 12 carbon atoms; R₃, R₄ and R₅ are hydrogen; n is a whole numberbetween 0 and 6; and the amidine group is in the para position relativeto the -A-NH-group, or a pharmaceutically acceptable salt thereof. 5.The compound according to claim 1 wherein A is a thiocarboxamide group;R₁ is an alkyl group having from 1 to 3 carbon atoms; R₂ is selectedfrom an aryl group, unsubstituted or substituted with halogen groups;R₃, R₄ and R₅ are hydrogen; n is a whole number between 0 and 6, and theamidine group is in the para position relative to the -A-NH-group, or apharmaceutically acceptable salt thereof.
 6. The compound according toclaim 1 wherein A is a thiocarboxamide group; R₁ is selectedindependently from a methyl group, or a nitro-amino group; R₂, R₃, R₄and R₅ are hydrogen; n is 5; and the amidine group is in the paraposition relative to the -A-NH-group.
 7. A pharmaceutical preparationcomprising a compound according to claim 1, or apharmaceutically-acceptable salt thereof, as an active ingredient and apharmaceutically acceptable carrier.
 8. The pharmaceutical preparationaccording to claim 7 further comprising pharmaceutically-acceptableinactive ingredients selected from the group consisting of vehicles,binders, flavourings, sweeteners, disaggregants, preservatives,humectants and mixtures thereof, ingredients which facilitate rectal,transdermal or transmucosal absorption, and ingredients that permit thecontrolled release of the active ingredient over time.
 9. A method forthe preparation of a compound of formula (I)

wherein A is selected independently from a carboxamide group, athiocarboxamide group, and a carbonyl group; R₁ is selected from analkyl group having from 1 to 3 carbon atoms and an amino groupsubstituted with a nitro group or a methyl group; R₂ is selectedindependently from hydrogen, an alkyl group having from 1 to 4 carbonatoms, a methoxy group, an ethoxy group, or a propoxy group, a mono-,bi- or tricyclic cycloalkane group having from 5 to 12 carbon atoms, anadamantyl group, and an aryl, naphthyl or heterocyclic group,unsubstituted or substituted with a methyl group, a methoxy group, ahydroxy group, an amino group or a halogen group; R₃ and R₄ are selectedindependently from hydrogen and an alkyl group having from 1 to 3 carbonatoms; R₅ represents one or two substituents independently selected fromhydrogen and a methyl group, a methoxyl group and a hydroxyl group; n isa whole number from 0 to 6; and an amidine group is in the para or metaposition relative to the -A-NH-group, or a pharmaceutically acceptablesalt thereof which comprises the steps of: a) reacting a 1,3 or1,4-phenylenediamine of formula (IV)

in which R₅ has the meaning given above, with the isothiocyanate offormula (V A), with the acyl chloride of formula (V B), or with theisocyanate of formula (V C),

in which R₂, R₃, and n have the meanings given above, with an excess ofphenylenediamine, in a neutral solvent and at a temperature of between4° C. and the reflux temperature of the solvent used, to give thecorresponding anilines of formula (III)

in which R₂, R₃ R₄, A and n have the meanings given above, and in whichthe amine group is in the meta or para position relative to the chainadjacent to the -A-NH-group; b) reacting the anilines of formula (III)with an appropriate imidate hydrochloride of formula (II)

in which R₁ has the meaning given above, in the presence of an excess offormula (II) and of the corresponding stoichiometric quantity of atertiary base, in an inert anhydrous solvent, at a temperature ofbetween 4° C. and the boiling point of the solvent to give thecorresponding derivatives of formula (I)